Gephyrin CRISPR Activation Plasmid (h): sc-401197-ACT

Human GPHN encodes gephyrin, a multifunctional scaffold that anchors inhibitory neurotransmitter receptors at postsynaptic membranes and organizes signaling nanodomains critical for synaptic transmission. Gephyrin binds and stabilizes GABA_A and glycine receptors and interfaces with cytoskeletal and trafficking components to regulate receptor clustering, synapse maturation, and plasticity. Beyond its synaptic role, gephyrin participates in molybdenum cofactor biosynthesis, linking it to essential redox enzyme activity and cellular metabolic homeostasis. Dysregulation of GPHN expression or gephyrin-dependent receptor organization has been associated with altered inhibitory circuit function and is relevant to neurodevelopmental and seizure-related phenotypes in mechanistic studies.

Gephyrin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GPHN expression without altering the underlying DNA sequence.

Gephyrin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GPHN locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the GPHN transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Gephyrin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GPHN locus and enabling the study of Gephyrin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Gephyrin pathway restoration in tumor cells with silenced or reduced GPHN expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.